Project Summary To mitigate the ongoing threat of anthrax infection due to bioterrorism, active military members are vaccinated with Anthrax Vaccine Adsorbed (AVA). However, some AVA recipients may remain unprotected against anthrax infection and a better understanding of mechanisms leading to impaired vaccine response and rapidly waning immunity are needed. In the largest real-world cohort of AVA vaccinees (>2,900 individuals), less than 50% of AVA vaccinees showed significant in vitro lethal toxin neutralization, and both antibody levels and neutralization capacity waned quickly after vaccination. Therefore, the goal of this project is to identify mechanisms of poor neutralization after AVA vaccination. The primary antigen in AVA is protective antigen (PA). Our previous studies have identified common sequential epitopes recognized by serum anti-PA, and have shown differential epitope binding of neutralizing vs. non-neutralizing responses. Poorly neutralizing responses have also been associated with impaired avidity of anti-PA and enhanced IgG4 production. In addition, suboptimal AVA responses are more common in African American vs. European American vaccinees. Preliminary data suggest that African American individuals have enriched responses against non-neutralizing epitopes after AVA vaccination and have marked differences in immune cell subsets and immune pathways compared to European American individuals. Critical questions remain in understanding the mechanisms of rapidly waning or impaired adult vaccination responses, such as those against AVA. This project addresses mechanisms of impaired protection after AVA immunization by comparing anti-PA antibodies from high and low neutralizers in the AVA cohort described above as well as new recruits for domain specificity, anti-PA avidity, and anti-PA IgG4 responses, all of which may inhibit protective human Bacillus anthracis immunity (Aim 1). Additional mechanisms of protective responses will be dissected using neutralizing PA-specific human monoclonal antibodies previously generated by our lab. In addition, although anti-PA domain specificity is related to neutralizing capacity, the conformational epitopes of PA bound by the serum of neutralizers have not been elucidated. Therefore, this project uses novel hydrogen-deuterium exchange mass spectrometry tech- niques to map anti-PA binding sites on PA using sera from high and low neutralizers (Aim 2). Finally, mech- anisms of impaired AVA responses will be evaluated in African American vs. European American vaccinees (Aim 3). Antibodies, immune cell profiles, and regulatory pathways will be compared by mass cytometry (CyTOF), intracellular cytokine production, flow cytometry and ELISpot at various times after AVA vaccination. Epitope specificity and genetic predisposition to impaired anti-PA responses will be tested. These studies will provide new insights to optimize future anthrax vaccines and other adult vaccinations across racial groups.